The ultimate objective of this research is to understand cobalamin- dependent propanediol metabolism in the enteric bacterium Salmonella typhimurium as a step towards controlling this organism's ability to cause disease. The five specific aims of this research plan are to: (1) identify all of the genes that are directly involved in propanediol metabolism, (2) define the genetic organization of the propanediol genes on the S typhimurium chromosome, (3) determine the physical structure of the cloned propanediol genes, (4) investigate the regulation of the propanediol genes, and (5) express the cloned genes that encode the cobalamin-dependent enzyme propanediol dehydratase. S typhimurium is a food-borne pathogen that causes acute gastroenteritis. Salmonellosis represents both a major public health problem and a serious economic burden in the United States. Propanediol metabolism is an important part of the anaerobic metabolism of S typhimurium and is likely to enhance its survival in the O2-free environment of the intestinal tract. Thus, this study will yield new and valuable information that may be used to find better ways of decreasing the incidence of this infection. The research design and methods have been crafted to achieve the specific aims of this project. These methods are to: (1) isolate and characterize mutants with defects in each and every enzymatic function that directly contributes to propanediol metabolism, (2) build a fine structure genetic (deletion) map of the propanediol genes and perform complementation tests with them, (3) build a physical (restriction) map of the cloned genes and determine their DNA sequence, (4) use in vivo genetic fusions to monitor changes in gene expression in cells grown under a variety of culture conditions, and (5) use the T7 RNA polymerase/promoter system to express the cloned propanediol dehydratase genes and analyze the resulting proteins by gel electrophoresis. This experimental approach emphasizes classical genetic analysis and recombinant DNA technology but also draws upon the tools of biochemistry and microbial physiology.